The hallmark of a vertebrate immune system is the ability to discriminate “self” from “non-self” (foreign). This property has led to the evolution of a system requiring multiple signals to achieve optimal immune activation (Janeway, Cold Spring Harbor Symp. Quant. Biol. 54:1-14 (1989)). T cell-B cell interactions are essential to the immune response. Levels of many cohesive molecules found on T cells and B cells increase during an immune response (Springer et al., A. Rev. Immunol. 5:223-252 (1987); Shaw and Shimuzu, Current Opinion in Immunology, Eds. Kindt and Long, 1:92-97 (1988)); and Hemler Immunology Today 9:109-113 (1988)). Increased levels of these molecules may help explain why activated B cells are more effective at stimulating antigen-specific T cell proliferation than are resting B cells (Kaiuchi et al., J. Immunol. 131:109-114 (1983); Kreiger et al., J. Immunol. 135:2937-2945 (1985); McKenzie, J. Immunol. 141:2907-2911 (1988); and Hawrylowicz and Unanue, J. Immunol. 141:4083-4088 (1988)).
The generation of a T lymphocyte (“T cell”) immune response is a complex process involving cell-cell interactions (Springer et al., A. Rev. Immunol. 5:223-252 (1987)), particularly between T and accessory cells such as B cells, and production of soluble immune mediators (cytokines or lymphokines) (Dinarello and Mier, New Engl. Jour. Med. 317:940-945 (1987)). This response is regulated by several T-cell surface receptors, including the T-cell receptor complex (Weiss et al., Ann. Rev. Immunol. 4:593-619 (1986)) and other “accessory” surface molecules (Springer et al., (1987) supra). Many of these accessory molecules are naturally occurring cell surface differentiation (CD) antigens defined by the reactivity of monoclonal antibodies on the surface of cells (McMichael, Ed., Leukocyte Typing III, Oxford Univ. Press, Oxford, N.Y. (1987)).
In order to achieve effective T lymphocyte activation, two receptors on the cell surface must be engaged by their respective ligands and deliver a signal to the cell. First the T cell receptor must recognize antigen in the context of MHC on an antigen presenting cell. Second, a co-stimulatory receptor must bind the appropriate ligand, or co-receptor, on the antigen presenting cell. The most studied T cell co-stimulatory receptor is CD28, which binds to B7 molecules (CD80 and CD86) on antigen presenting cells. Green J L, Leytze G M, Emswiler J, Peach R, Bajorath J, Cosand W, Linsley P S. Covalent dimerization of CD28/CTLA-4 and oligomerization of CD80/CD86 regulate T cell costimulatory interactions. J. of Biol. Chem. 271: 26762-26771, 1994. Inhibition of the CD28/B7 pathway in vitro inhibits T cell proliferation, cytokine production and induces antigen specific T cell unresponsiveness. Green J L, Leytze G M, Emswiler J, Peach R, Bajorath J, Cosand W, Linsley P S. Covalent dimerization of CD28/CTLA-4 and oligomerization of CD80/CD86 regulate T cell costimulatory interactions. J. of Biol. Chem. 271: 26762-26771, 1994; and Kelly S, Linsley P, Warner G, Shyu W C and Paborji M. Investigator Brochure, BMS-188667, CTLA4Ig. In animal models, this pathway has been shown to be important in T cell-dependent immune responses, including alloantigen recognition and autoimmunity. Green J L, Leytze G M, Emswiler J, Peach R, Bajorath J, Cosand W, Linsley P S. Covalent dimerization of CD28/CTLA-4 and oligomerization of CD80/CD86 regulate T cell costimulatory interactions. J. of Biol. Chem. 271: 26762-26771, 1994; and Kelly S, Linsley P, Warner G, Shyu W C and Paborji M. Investigator Brochure, BMS-188667, CTLA4Ig. Larsen, C. P., Pearson, T. C., Adams, A. B., Tso, P., Shirasugi, N., Strobert, E., Anderson, D., Cowan, S., Price, K., Naemura, J., Emswiler, J., Greene, J., Turk, L., Bajorath, J., Townsend, R., Hagerty, D., Linsley, P. S., and R. J. Peach. 2005. Rational Development of LEA29Y, a High-Affinity Variant of CTLA4-Ig with Potent Immunosuppressive Properties. American Journal of Transplantation. 5(3):443-53. Thus, the CD28/B7 pathway represents a viable, logical target for an immunomodulatory therapeutic agent.
CTLA4Ig (BMS-188667), a fusion protein comprising the extracellular domain of human CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) and a fragment of the Fc domain of human IgG1, blocks the CD28/B7 pathway by binding to CD80 and CD86 on the surface of the antigen presenting cells. This compound has been found to be clinically useful as an immunosuppressant. See U.S. patent application Ser. No. 10/419,008 (Publication No. 20040022787 A1), hereby incorporated by reference in its entirety, which describes and discusses CTLA4Ig and L104EA29YIg and methods of preparation and use thereof U.S. Pat. Nos. 5,844,095, 5,885,796, and 5,851,795, also incorporated by reference in their entirety, describe and discuss CTLA4Ig.
A related molecule, L104EA29YIg (BMS-224818) (also known as LEA29Y), was found to be a particularly potent immunomodulatory therapeutic agent. This compound is a human CTLA4Ig molecule containing a two amino acid substitution that results in enhanced binding to CD80 and CD86 relative to CTLA4Ig. See Larsen, C. P., Pearson, T. C., Adams, A. B., Tso, P., Shirasugi, N., Strobert, E., Anderson, D., Cowan, S., Price, K., NaemurFfia, J., Emswiler, J., Greene, J., Turk, L., Bajorath, J., Townsend, R., Hagerty, D., Linsley, P. S., and R. J. Peach. 2005. Rational Development of LEA29Y, a High-Affinity Variant of CTLA4-Ig with Potent Immunosuppressive Properties. American Journal of Transplantation. 5(3):443-53. U.S. patent application Ser. No. 09/865,321 (Publication No. 2002-0182211 A1), which is also hereby incorporated by reference in its entirety, describes and discusses L104EA29YIg.
CD80 and CD86 are discussed in Carreno, B. M., and Collins, M., 2002 (The B7 Family of Ligants and Its Receptors: New Pathways for Costimulation and Inhibition of Immune Responses, Annu. Rev Immunol. 20:29-53) and Salomon, B., and Bluestone, J. A., 2001 (Complexities of CD28/B7: CTLA-4 Costimulatory Pathways in Autoimmunity and Transplantation, Annu. Rev. Immunol. 19:225-52). Given that CTLA4Ig and L104EA29YIg bind to circulating leucocytes expressing CD80 and/or CD86 molecules, it would be informative to monitor the extent to which CD80 and/or CD86 is bound to the fusion protein(s), in addition to the amount of compound circulating in the plasma during clinical use. In doing so, clinicians would be able to correlate compound exposure levels with receptor saturation levels required for efficacy in order to monitor binding efficiency. Understanding the extent to which CD86 is saturated with L104EA29YIg at various blood concentrations can be used to help justify different dosing schemes or regimes. For example, during the development phase, different formulations and routes of administration will be utilized (e.g. monthly intravenous or weekly subcutaneous treatment). This assay could be used to help establish the best route and course of administration which demonstrates maximum saturation for the longest period of time.